Notch Signaling
Notch signaling is a mechanism conserved through evolution and plays a role in determining cell fate choices during the development of many cell lineages in both vertebrates and invertebrates. The cellular responses to Notch signaling activation that have been characterized so far are, for example, differentiation, proliferation, and/or apoptosis, depending on the specific point in the cell cycle and the specific cell type. In addition to its role as a signal-transducing cell surface protein, Notch can also directly regulate gene transcription.
Four mammalian Notch receptors (Notch-1, Notch-2, Notch-3, and Notch-4) and five Notch ligands (delta-like ligands-1, -3, and -4 and Jagged-1 and -2) have been identified (Artavanis-Tsakonas, et al., Science, 284:770-776 (1999)). The Notch receptors are typically large (generally about 300 kDa) single pass transmembrane receptors initially identified in the fruit fly (the phenotype of the Notch-1 mutant allele has notched wings). The Notch receptors have an extracellular domain with numerous (usually over 30) EGF-like repeats, three membrane-proximal Notch-specific repeats, and an intracellular domain that contains four functional regions. These four intracellular functional regions are the RAM domain, the 6 ankyrin repeats, a transcriptional factor activation domain (TAD), and the proline, glutamate, serine, and threonine-rich PEST sequence. Also, there are two nuclear localization sequences both before and after the ankyrin repeats.
Typically, Notch receptors are synthesized in the endoplasmic reticulum and are then cleaved in an established three step proteolytic model of Notch activation. The initial cleavage results from activation of a furin-like convertase in the trans-Golgi network and occurs at site 1, between the Notch EGF repeats and the transmembrane domain. The resulting halves re-associate via a calcium-dependant, non-covalent bond (Rand, et al., Mol Cell Biol, 20:1825-1835 (2000)). This heterodimer migrates to the cell surface where cell-membrane-associated ligands on the adjacent cell bind to and activate the Notch receptors. Activation of the Notch receptor by these ligands triggers the second cleavage of the extracellular portion of the Notch receptor by the metalloprotease TACE (TNF-α-converting enzyme). This is followed by the cleavage of Notch from the membrane by the presenilin-dependent γ-secretase complex, which releases the cytosolic fragment known as the Notch intracellular domain (NICD) (Fortini, Nature Rev, 3:673-684 (2002)).
NICD is approximately 80 kDa in length and binds to a number of transcription factors, including RBP-Jκ, which together with NICD forms a complex that promotes transcription of the Hairy Enhancer of Split (HES) (Jarriault, et al., Mol Cell Biol, 18:7423-7431 (1998)), HERP, and HEY gene families. HES acts a transcriptional factor repressor of, amongst others, genes associated with stem cells of the gut such as ngn3 (Lee, et al., Diabetes, 50(5):928-936 (2001)) and Math1 (Yang, et al., Science, 294(5549):2155-2158 (2001)). Thus, to date the Notch signaling pathway is known to comprise the Notch ligands delta-like ligands-1, -3, and -4 and Jagged-1 and -2; the Notch receptors Notch-1 through Notch-4; intracellular effectors CBF-1, Deltex, and NF-κB; the Notch target genes HES, HERP, HEY, and bHLH; processing molecules Kuzbanian, TACE, sel1L, and presenilin; and factors known to regulate Notch pathway activity such as numb, numb-like, and disheveled-1, -2, and -3, as well as fringe family members lunatic fringe, manic fringe, and radical fringe.
Diabetes and the Pancreas
Blood glucose levels are controlled by the release of insulin from the β-cells of the islets of Langerhans in the endocrine portion of the pancreas. Diabetes is basically a lack of sufficient functional β-cell mass to control increases in blood glucose. There are two main categories of diabetes: type 1 (commonly referred to as childhood diabetes) in which there are no β-cells and type 2 (or late onset diabetes) in which insulin secretion is altered and results from a failure of the β-cells to produce sufficient insulin to meet the demands of the body.
Currently, nearly 18.2 million people (6.3% of the population) in the US have diabetes. Of those, 13 million carry the diagnoses but nearly a third (5.2 million) have not been diagnosed. Of those Americans that have been diagnosed with diabetes, approximately 5-10% suffer from type-1 diabetes and 90-95% suffer from type-2 diabetes. Diabetes presently costs $132 billion in health care expenditure in the US alone (Diabetes Care, Economic Costs of Diabetes in the U.S. 2002-2003, 26: 917-932).
Type-1 diabetes results primarily from an autoimmune destruction of the β-cells that secrete insulin. Thus, there is usually a complete deficiency of insulin in this state and patients are required to take insulin injections to survive. Patients suffering from type-2 diabetes have abnormal insulin secretion and therefore may not be dependent on insulin unless control of blood glucose is not achieved by diet and exercise and oral hypoglycemic agents. Medications currently in use to treat type-2 diabetes have limited success in controlling blood glucose levels, hence, the complications of the disease (United Kingdom Prospective Diabetes Study, 1998). An ongoing failure in β-cell function is another aspect of this disease that none of the currently used medications is capable of reversing (United Kingdom Prospective Diabetes Study, 1995).
There is a third form of diabetes referred to as maturity onset diabetes of the young or MODY. So called as its onset has many of the classical symptoms associated with type-2 diabetes but occurs in the younger population range where the average age at which symptoms present is usually before 25 years of age (Fajans, et al., N Eng J Med, 13:971-980 (2001)). The symptoms of MODY are associated with disrupted expression of genes for proteins that are essential for the maintenance of β-cell function and mass. There are currently 6 defined MODY types, MODY-1, -2, -3, -4, -5, and -6, connected with HNF-4α, glucokinase, HNF-1α, PDX-1, HNF-1β, and, β2/NeuroD genes, respectively.
Insulin is secreted in exquisitely controlled amounts in response to increases in blood glucose. Current medications that affect, β-cells and insulin secretion directly are the class of drugs which act as potassium channel closers; these drugs are divided into two groups, the sulfonylureas and the benzoic acid derivatives (also referred to as meglitinides). These drugs act to depolarize the membrane potential of the β-cell by closing the KATP channels, thereby increasing the release of insulin secretory granules from the ready releasable pool. They do not increase insulin synthesis nor do they improve the responsiveness of the β-cell to rising blood glucose levels. Glucagon-like-peptide-1 (GLP-1) receptor analogs have been shown to increase insulin secretion, insulin synthesis and to sensitize the response of the β-cells to changes in blood glucose. (Drucker D. J. Glucagon-like peptide-1 and the islet, β-cell; augmentation of cell proliferation and inhibition of apoptosis. Endocrinology 144:5145-5148, (2003)). They improve insulin secretion by acting at several points of the insulin secretion pathway. GLP-1 receptor activation is currently the only pharmacological agent that increases insulin transcription and translation, i.e., enhances insulin synthesis and secretion in the β-cell. It is also the only compound which apparently improves long term function of the β-cell as indicated by a restoration of first phase insulin secretion in patients with type-2 diabetes.
In light of the prevalence and seriousness of diabetes, as well as other metabolic disorders, there exists a need for new diagnostic, therapeutic, and preventative processes and compositions. The disclosed subject matter addresses this need.